Journal: Journal of Biological Chemistry

Article Title: MicroRNA-223-3p inhibits vascular calcification and the osteogenic switch of vascular smooth muscle cells

doi: 10.1016/j.jbc.2021.100483

Figure Lengend Snippet: Figure 6. STAT3 is a potential miR-223-3p target in vascular calcification. A, MiR-223-3p target sites (in red letters) in the STAT3 30 UTRs. B, STAT3 expression in primary vascular smooth muscle cells (mVSMCs) transfected with miR-223-3p or a control mimic (n = 3 per group). C–E, representative western blot and quantification of STAT3 expression in mVSMCs (C), MOVAS (D), and 3T3 cells (E) transfected with miR-223-3p or a control mimic (n = 3 per group). F–G, Representative images (E) and quantification (F) of STAT3 IHC in the brachiocephalic trunks of aged ApoE KO and DKO mice (n = 6 per group). Data are expressed as the mean ± SD. *p <0.05 by unpaired two-tailed Student’s t-test.

Article Snippet: Inhibition of STAT3 expression by siRNA The siRNA against mouse STAT3 and scrambled siRNA were purchased from Santa Cruz Biotechnology, Inc (TX, USA).

Techniques: Expressing, Transfection, Control, Western Blot, Two Tailed Test

Journal: Journal of Biological Chemistry

Article Title: MicroRNA-223-3p inhibits vascular calcification and the osteogenic switch of vascular smooth muscle cells

doi: 10.1016/j.jbc.2021.100483

Figure Lengend Snippet: Figure 7. IL-6-induced VSMC calcification is blocked by miR-223-3p. A, RT-PCR analysis of osteogenic genes in VSMCs. VSMCs were cultured in oste- ogenic media (OIM) with 50 ng/ml mIL-6/sIL-6R complex or PBS for 10 days (n = 3). B, effects of miR-223-3p on osteogenic gene expression in VSMCs. VSMCs were transfected with 100 nM miR-223-3p or a control mimic and cultured in osteogenic medium (n = 3). C, representative western blot and quantification of STAT3 expression in VSMCs cultured as in B for 14 days (n = 4 per group). D, representative images of Alizarin Red S staining of VSMCs cultured as in B for 21 days. E, total calcium content in VSMCs cultured as in B for 14 days (n = 3). F, representative western blot of STAT3 in VSMCs transfected with vector (Lenti-Ctrl) or lentivirus packaged with STAT3(Lenti-Stat3). G, representative images of ARS staining of VSMCs. VSMCs transfected with Lenti-Ctrl or Lenti- Stat3 were cultured in osteogenic media with 50 ng/ml mIL-6/sIL-6R complex for 21 days, and miR-223-3p or a control mimic(100 nM) was transfected in indicated wells. H, total calcium content in VSMCs cultured as in G for 14 days (n = 3). Data are expressed as the mean ± SD. *p <0.05, **p <0.01 compared with the first group, #p <0.05 compared with the second group. Two groups were assessed by unpaired two-tailed Student’s t-test. For comparisons of more than two groups, one-way analysis of variance was used.

Article Snippet: Inhibition of STAT3 expression by siRNA The siRNA against mouse STAT3 and scrambled siRNA were purchased from Santa Cruz Biotechnology, Inc (TX, USA).

Techniques: Reverse Transcription Polymerase Chain Reaction, Cell Culture, Gene Expression, Transfection, Control, Western Blot, Expressing, Staining, Plasmid Preparation, Two Tailed Test

Journal: Redox biology

Article Title: Metformin inhibits methylglyoxal-induced retinal pigment epithelial cell death and retinopathy via AMPK-dependent mechanisms: Reversing mitochondrial dysfunction and upregulating glyoxalase 1.

doi: 10.1016/j.redox.2023.102786

Figure Lengend Snippet: Fig. 5. AMPK activator A769662 mimics the effect of metformin in MGO-stimulated cells (A) Cells were pretreated with metformin (6 mM) or A769662 (25 μM) 30 min prior to MGO (300 μg/ml) treatment for indicated time points. AMPK phosphorylation was measured by immunoblotting. (B) Cells were 30 min pretreated with A769662 (25 μM) prior to MGO (300 μg/ml) for 6 h. Cell viability was measured by Annexin V-FITC/PI using FACS. (C, D, E, F) Cells were pretreated with A769662 (25 μM) for 30 min followed by MGO (300 μg/ml) treatment for 4 h. DCFDA (C), DHE (D), mitoSOX (E), and mitoPY1 (F) were used to measure cellular ROS. (G) Cells were 30 min pretreated with compound C (10 μM) followed by MGO (300 μg/ml) stimulation for 6 h. In some experiments, ARPE-19 cells were treated with siRNA followed by stimulation with MGO (300 μg/ml) for 6 h. Cell viability was measured by Annexin V-FITC/PI using FACS. AMPK expression after siRNA treatment was determined by immunoblotting. (H) Immediately after treatment with A769662 (25 μM), cells were subjected into SFe24 analyzer for OCR measurement, as described in Fig. 3D. Data were the mean ± S.E.M. from at least 3 independent experiments. *p < 0.05, indicating the significant effect of MGO; #p < 0.05, indicating the significant effects of A769662, compound C and AMPK silencing on MGO- induced responses.

Article Snippet: Human si-AMPKα1/2 (sc-45312) and scramble nonspecific siRNA (sc-44236) were purchased from Santa Cruz Biotechnology (Dallas, Texas, USA) and human si-GLO1 (s5825) was from Thermo Fischer Scientific (Waltham, US).

Techniques: Phospho-proteomics, Western Blot, Expressing

Journal: Redox biology

Article Title: Metformin inhibits methylglyoxal-induced retinal pigment epithelial cell death and retinopathy via AMPK-dependent mechanisms: Reversing mitochondrial dysfunction and upregulating glyoxalase 1.

doi: 10.1016/j.redox.2023.102786

Figure Lengend Snippet: Fig. 8. Metformin and A769662 reverse MGO-induced GLO1 downregulation. (A) Cells were pretreated with BBGC (10 μM), metformin (6 mM) and/or A769662 (25 μM) 30 min before MGO (100 μg/ml) stimulation. After 4 h, cell viability was determined by Annexin V-FITC/PI staining using FACS. (B) Cells were treated with siRNA to silence GLO1, then treated with MGO (100 or 300 μg/ml) for 6 h. Cell viability was determined by Annexin V-FITC/PI staining using FACS. (C–E) Cells were pretreated with metformin (6 mM) and/or A769662 (25 μM) 30 min prior to MGO (300 μg/ml) stimulation. (C) After incubation for 1, 3, or 6 h, cell lysates were prepared for immunoblotting. (D) After incubation for 2 or 4 h, GLO1 gene expression was measured using PCR analysis. (E) After incubation for 3 or 6 h, GLO1 and GLO2 activities were determined by commercial kits according to the manufacturer’s instructions. Data were the mean ± S.E.M. from at least 3 independent experiments. *p < 0.05, indicating the significant effect of MGO. #p < 0.05, indicating the blockade effects of metformin and A769662.

Article Snippet: Human si-AMPKα1/2 (sc-45312) and scramble nonspecific siRNA (sc-44236) were purchased from Santa Cruz Biotechnology (Dallas, Texas, USA) and human si-GLO1 (s5825) was from Thermo Fischer Scientific (Waltham, US).

Techniques: Staining, Incubation, Western Blot, Gene Expression